The long-term goal of this project is to characterize neurotransmitter receptor-mediated information transduction, and its regulation, across neuronal membranes. The primary receptor systems under investigation are those for the neurotransmitter dopamine. To characterize these receptors at the biochemical and molecular levels and study their regulation, two interrelated lines of research are underway: 1) investigation of the cell biology, structure, function and regulation of the receptors at the protein level; and 2) the molecular cloning and identification of proteins that directly interact with the receptors to modify their function, expression, regulation and trafficking. Projects involing mice that are deficient in specific dopamine receptor subtypes are also in progress. In FY2004, we have investigated the role of protein kinase C (PKC)-mediated phosphorylation in regulating both D1 and D2 receptor function. Previously, D2 dopamine receptors (D2 DARs) have been shown to undergo G protein-coupled receptor kinase (GRK) phosphorylation in an agonist-specific fashion. We have now investigated the ability of the second-messenger activated protein kinases, protein kinase A (PKA) and protein kinase C (PKC) to mediate phosphorylation and desensitization of the D2 DAR. HEK293T cells were transiently transfected with the D2 DAR and then treated with intracellular activators and inhibitors of PKA or PKC. Treatment with agents that increase cAMP, and activate PKA, had no effect on the phosphorylation state of the D2 DAR suggesting that PKA does not phosphorylate the D2 DAR in HEK293T cells. In contrast, cellular treatment with phorbol 12-myristate 13-acetate (PMA), a PKC activator, resulted in ~3-fold increase in D2 DAR phosphorylation. The phosphorylation was specific for PKC as the PMA effect was mimicked by PDBu, but not by 4alphaPDD, active and inactive, phorbol diesters, respectively. The PMA-mediated D2 DAR phosphorylation was completely blocked by co-treatment with the PKC inhibitor, bisindolylmaleimide II and augmented by co-transfection with PKC betaI. In contrast, PKC inhibition had no effect on agonist-promoted phosphorylation suggesting that PKC is not involved in this response. PKC phosphorylation of the D2 DAR was found to promote receptor desensitization as reflected by a decrease in agonist potency for inhibiting cAMP accumulation. Interestingly, PKC phosphorylation also promoted internalization of the D2 DAR through a beta-arrestin- and dynamin-dependent pathway; a response not usually associated with PKC phosphorylation of GPCRs. Site-directed mutagenesis experiments resulted in the identification of two domains of PKC phosphorylation sites within the 3rd intracellular loop of the receptor. Both of these domains are involved in regulating sequestration of the D2 DAR whereas only one domain is involved in receptor desensitization. These results indicate that PKC can mediate phosphorylation of the D2 DAR resulting in both functional desensitization and receptor internalization. The D1 dopamine receptor (D1) also contains several consensus sequences for PKC-mediated phosphorylation, yet the role of PKC in D1 signaling remains largely unexplored. In FY2004, we have investigated PKC-mediated phosphorylation of D1 using transiently transfected HEK293T cells and metabolic labeling assays. Activation of PKC through cellular treatment with the phorbol ester PDBu produces an increase in the phosphorylation state of D1. This effect is mimicked by the PKC activator PMA, but not by the inactive phorbol ester 4alphaPDD. PDBu-stimulated D1 phosphorylation is both time and dose dependent achieving maximal results after 30 min of treatment with 1 microM of PDBu. Interestingly, the phosphorylation is transient in nature returning to basal levels after 60 min of treatment despite the continued presence of PDBu. Both basal and PDBu-mediated D1 phosphorylation can be blocked with cell permeable PKC inhibitors. Co-transfection of the cells with PKC isoforms beta, delta, epsilon and lambda resulted in greater D1 phosphorylation, whereas PKC isoforms mu and zeta were without effect. Further, co-transfection with the Gq-linked M1 muscarinic receptor resulted in increased D1 phosphorylation upon treatment with the muscarinic agonist carbachol, indicating that D1 can be phosphorylated by PKC in a heterologous fashion. The functional effects of PKC activation on D1 signaling were evaluated using intact cell cAMP accumulation assays. Surprisingly, PDBu treatment was found to potentiate dopamine-stimulated cAMP accumulation mediated by D1. This effect was mimicked by PMA, but not by 4alphaPDD, and was attenuated by PKC inhibitors, indicating the involvement of PKC. The potentiation is specific to D1 activation, since pretreatment of cells with PDBu had no effect on forskolin-stimulated cAMP accumulation. These data suggest that PKC plays a multi-faceted role in D1 signaling where PKC phosphorylation of D1, in the absence of dopamine, may regulate the ?tone? or signaling potential of the receptor. In FY2004, we also continued our proteomics work directed at identifying interacting proteins for dopamine receptor subtypes. In order to identify interacting proteins for the rat D2 dopamine receptors, we have used the DupLex-A yeast two-hybrid system. The entire third cytoplasmic loop of rat D2 was used as a bait to identify clones encoding interacting proteins from a rat whole brain cDNA library. This resulted in the identification of several positive clones. Sequence analysis indicated that two clones were identical and sequences encoded amino acid residues (167-439) of PKC zeta interacting protein-1 (ZIP-1). The specificity of the interaction of the D2 receptor and the ZIP-1 clone was verified using a corresponding yeast expression vector that lacks inserts, and showed no growth on -leu plates and no color change on X-gal plates. The ZIP-1 clone was further shown not to interact with the third cytoplasmic loops of the rat D1, D3, D4, and D5 dopamine receptors. However, the ZIP-1 clone interacted with both the D2S and D2L dopamine receptor isoforms. Two additional splice variants exist for ZIP (ZIP-2 and ZIP-3) and we found that the D2 receptor interacts with both. ZIP-3 is truncated at residue 234 indicating that the receptor interaction domain is within residues 167-234. Biochemical verification of this interaction was obtained by co-expressing epitope-tagged D2 dopamine receptor and full-length ZIP-1 in HEK293T cells and showing that both proteins could be co-immunoprecipitated from solubilized cells. Co-expression of D2-YFP and c-Myc tagged ZIP-1 in HEKT cells also demonstrated co-localization using immunohistochemical and fluorescence techniques. Co-expression of the ZIP-1 cDNA with the D2 dopamine receptor resulted in a 60% reduction of receptor binding activity. In contrast, there was no effect on D2 receptor-mediated inhibition of intracellular cAMP accumulation. Further experiments characterizing the interactions between ZIP-1 and the D2 receptor are in progress. In FY2004, we initiated a new proteomics project involving a co-immunoprecipitation (co-IP) assay for D1 DARs coupled with mass spectrometry (MS) sequencing to identify interacting partners. Following IP of the D1 DAR from HEK293 cells, proteins were separated using 1D PAGE. Iindependent bands were excised, trypsinized, and subjected to MS-based peptide sequencing. Approximately 40 proteins were identified by searching against a non-redundant protein database. Approximately, 14 of these proteins were also found in immunoprecipitates from mock-transfected controls and therefore identified as non-specific. These findings demonstrate that the D1 DAR is expressed as part of a protein complex that is capable of being selectively isolated via co-immunoprecipitation assays.